The long-term goal of the proposed research is to better understand molecular mechanisms in the regulated production of ribosomal and messenger RNA. Huge advances have been made in the identification of proteins involved in transcription and RNA processing for RNA polymerase I and II, yet there are many important questions to be answered concerning basic mechanisms. Our unique approach offers a means to answer some of these questions by direct visualization of active genes using the Miller chromatin spreading method for electron microscopy. In brief, using Saccharomyces cerevisiae as a model system, we visualize specific genes in the presence and absence of key regulatory proteins (i.e., in appropriate mutants) to determine how the gene is changed in its absence. We are able to discern (and distinguish) effects on transcription initiation, elongation, termination, processing of nascent transcripts, chromatin structure, and template topology. Furthermore, for each of these topics, we are able to resolve patterns that are informative regarding the molecular mechanism affected. Our recent results have led us to focus on the following Aims: (Aim 1) Determine the role of histones and chromatin-associated elongation factors in elongation by RNA polymerase I; (Aim 2) Analyze topological abnormalities in rDNA that appear in the absence of topoisomerases and ask if and how these correlate with the increased recombination that occurs in these mutants; and (Aim 3) Develop and apply a system for the visualization of specific Pol II genes and analysis of co-transcriptional events. These studies are relevant to human health in the following ways: we will gain information on the regulation of ribosome production, which is important to cell growth control and is up- regulated in cancer; (b) we will gain information about the mechanisms of transcription elongation and splicing, mutations in which lead to many different human diseases, and (c) we may gain direct information on mechanisms of genomic instability by studying this process in topoisomerase-deficient cells.